Differentiation Process Of Stem Cells Research Articles

Monitoring Endoplasmic Reticulum Peroxynitrite Fluctuations in Primary Tendon-Derived Stem Cells and Insights into Tendinopathy.

Tendinopathy is one of the most prevalent musculoskeletal disorders, significantly affecting the quality of life of patients. Treatment outcomes can be improved with an early diagnosis and timely targeted interventions. Increasing evidence indicates that ROS and endoplasmic reticulum (ER) stress play key roles in modulating the differentiation processes of tendon-derived stem cells (TDSCs), thereby contributing to the initiation and progression of tendinopathy. However, the relationship between ONOO- and the differentiation process, as well as the various stages of tendinopathy, remains unexplored. Herein, we developed two highly specific and sensitive fluorescent probes (Rod-Cl and Rod-Br) for detecting ONOO- in the ER. Rod-Br can detect basal levels of ONOO- in the ER of TDSCs and measure ONOO- levels in primary TDSCs stimulated by interleukin-1β over various durations, allowing for comparisons between chondrogenic and osteogenic differentiation and ER stress levels. Additionally, we examined ONOO- variations in different stages of tendinopathy and treatment rat models in vivo and discussed the potential mechanisms. This research provides a robust tool for analyzing ONOO- dynamics in the tenogenic and osteogenic differentiation of TDSCs, offering new insights into the pathophysiology and treatment of tendinopathy.

Intratympanic injections made from eutectic liquid (camphor and menthol)-based oil-less emulsions containing morin hydrate-lipoid E80 complex: An approach to augment drug permeation into inner ear region in a healthy rabbit model

Morin hydrate (MH, flavonols of plants, family-Moraceae) promotes inner ear stem cell survival and differentiation process to preserve the healthy hearing function. However, round window membrane (RWM) of inner ear provides a barrier for MH to reach an adequate amount at inner ear region. Since MH possesses a low aqueous solubility (28.72 ± 0.97 μg/ml), intratympanic (IT) injections of camphor-menthol eutectic liquid-based oil-less emulsions containing MH-Lipoid E80 complex was developed to promote MH ferrying action across RWM. At 360 min post-dissolution time point, the MH solution displayed 92.88 ± 6.26 % drug release, but the drug-Lipoid E80 complex-loaded oil-less emulsions showed 58.05 ± 11.62 % only. The MTT assay results indicate the possible non-toxicity of oil-less emulsions to L-929 cells up to 100 μg/ml concentration. Significant differences were seen between the Cmax and AUC0-t values observed at 90 min in the perilymph after the IT injections of MH solution (14.13 ± 0.33 × 103 ng/ml & 429.55 ± 9.20 × 103 ng min/ml) and MH-Lipoid E80 complex-loaded oil-less emulsions (117.93 ± 4.24 × 103 ng/ml & 5529.46 ± 37.65 × 103 ng min/ml) into the healthy albino rabbits. Hence the IT injections of eutectic liquid-based oil-less emulsions could facilitate the drugs crossing over of RWM in the inner ear regions.

Kynurenine’s Effect on the Different Stages of Osteoblastic Differentiation

This study focuses on the dynamics of age-related changes in osteoblasts’ matrix production, with a specific focus on the involvement of the aryl hydrocarbon receptor (AhR). The AhR is activated by kynurenine, a metabolite of the amino acid tryptophan that tends to increase with age. Kynurenine has been known to impair osteoblasts’ bone formation ability, however, the point at which this occurs is unknown. The study utilizes a 21-day differentiation process of mesenchymal stem cells. “Window experiments” that vary the intervals of kynurenine treatment to explore its impact at different stages of osteoblast development were utilized. The experimental setup involves four distinct conditions: a control group with a vehicle, a group treated with kynurenine, a group with an AhR antagonist (BAY), and a group treated with both kynurenine and BAY. The AhR antagonist, specifically BAY2416964, serves to investigate whether any observed effects of kynurenine are mediated through AhR signaling. Results, analyzed through alizarin red staining, demonstrate a notable negative impact of kynurenine on matrix production, particularly in the middle stages of osteoblast differentiation. The study further employs crystal violet staining to confirm the presence of cells in the experimental wells, providing assurance of the reliability of the alizarin staining procedure. Importantly, this staining procedure also suggests that the effects of kynurenine on mineralized matrix production are largely independent of any potential impacts on cell viability.

Kynurenine’s Effect on the Different Stages of Osteoblastic Differentiation

This study focuses on the dynamics of age-related changes in osteoblasts’ matrix production, with a specific focus on the involvement of the aryl hydrocarbon receptor (AhR). The AhR is activated by kynurenine, a metabolite of the amino acid tryptophan that tends to increase with age. Kynurenine has been known to impair osteoblasts’ bone formation ability, however, the point at which this occurs is unknown. The study utilizes a 21-day differentiation process of mesenchymal stem cells. “Window experiments” that vary the intervals of kynurenine treatment to explore its impact at different stages of osteoblast development were utilized. The experimental setup involves four distinct conditions: a control group with a vehicle, a group treated with kynurenine, a group with an AhR antagonist (BAY), and a group treated with both kynurenine and BAY. The AhR antagonist, specifically BAY2416964, serves to investigate whether any observed effects of kynurenine are mediated through AhR signaling. Results, analyzed through alizarin red staining, demonstrate a notable negative impact of kynurenine on matrix production, particularly in the middle stages of osteoblast differentiation. The study further employs crystal violet staining to confirm the presence of cells in the experimental wells, provid-ing assurance of the reliability of the alizarin staining procedure. Importantly, this staining procedure also suggests that the effects of kynurenine on mineralized matrix production are largely independent of any potential impacts on cell viability.

RNA modification in normal hematopoiesis and hematologic malignancies.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells. Previous studies have shown that m6A plays a critical role under both normal physiological and pathological conditions. Hematopoiesis and differentiation are highly regulated processes, and recent studies on m6A mRNA methylation have revealed how this modification controls cell fate in both normal and malignant hematopoietic states. However, despite these insights, a comprehensive understanding of its complex roles between normal hematopoietic development and malignant hematopoietic diseases remains elusive. This review first provides an overview of the components and biological functions of m6A modification regulators. Additionally, it highlights the origin, differentiation process, biological characteristics, and regulatory mechanisms of hematopoietic stem cells, as well as the features, immune properties, and self-renewal pathways of leukemia stem cells. Last, the article systematically reviews the latest research advancements on the roles and mechanisms of m6A regulatory factors in normal hematopoiesis and related malignant diseases. More importantly, this review explores how targeting m6A regulators and various signaling pathways could effectively intervene in the development of leukemia, providing new insights and potential therapeutic targets. Targeting m6A modification may hold promise for achieving more precise and effective leukemia treatments.

Construction of an interactome network among circRNA-miRNA-mRNA reveals new biomarkers in hBMSCs osteogenic differentiation

Human bone marrow mesenchymal stem cells (hBMSCs) are adult stem cells residing in the bone marrow, characterized by their capacity for multi-directional differentiation, self-renewal, migration, and engraftment. Serving as seed cells, BMSCs play a pivotal role in the regeneration of bone defects. Hence, investigating the transcription factors and signaling pathways involved in the regulation of osteogenic differentiation in BMSCs holds significant importance. Recent research has unveiled that certain circular RNAs (circRNAs) can function as molecular sponges, influencing the osteogenic differentiation process of mesenchymal stem cells. However, many circRNAs remain undiscovered, and their precise mechanisms remain elusive. Therefore, the objective of this study is to construct an osteogenic differentiation-related circRNA-miRNA-mRNA network in hBMSCs. Subsequently, through bioinformatics analysis, we constructed a ceRNA network related to the osteogenic differentiation ability of hBMSCs, comprising 22 circRNAs, 17 miRNAs, and 15 mRNAs. The potential circRNA-miRNA-mRNA axes, including the role of hsa_circ_0001600 in promoting the osteogenic differentiation of hBMSCs through the targeted regulation of hsa-miR-542-3p, were validated through in vitro experiments.

IL-4 promotes chondrogenesis of bone marrow mesenchymal stem cellsand blockade of IL-4Rα retards the endochondral ossification during rat embryonic bone development.

Interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signalling pathways play important roles in the complex process of bone formation and bone remodelling. However, whether IL-4/IL-4Rα participates in skeletogenesis during embryonic development is not completely understood. We used the anti-IL-4Rα monoclonal antibody (anti-IL-4Rα mAb) as a powerful investigational tool to evaluate the potential roles of IL-4/IL-4Rα in the chondrogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Simultaneously, we explored the effect of IL-4/IL-4Rα on bone ossification during rat embryo-fetal development. In this study, we found that, compared to the control group, IL-4 can significantly promote the chondrogenic differentiation of BMSCs. Furthermore, following exposure to anti-IL-4Rα mAb in pregnant rats, unexpected phenomena were observed in fetal bone development, including non-ossification of the fetal sternum, an incomplete ossification centre in long bones and a reduced number of ossification points in digit (toe) bones. To further investigate the underlying mechanism of the phenotype, we studied the rat sternum as the target organ, starting from different time points of sternum development in the embryonic stage. The results indicated that the retardation mainly occurred in the middle and late stages of embryonic development. This retardation was characterized by the inhibition of the differentiation process of mesenchymal stem cells into chondrocytes, resulting in reduced angiogenesis near the ossification centre, failure of osteoblasts to invade the centre of the cartilage body with the blood vessels and delayed formation of the primary ossification centre (POC). Overall, our study demonstrated the significant function of IL-4/IL-4Rα in chondrogenic differentiation of BMSCs and bone ossification during embryo-fetal development.

Tumor suppressor miR-317 and lncRNA Peony are expressed from a polycistronic non-coding RNA locus that regulates germline differentiation and testis morphology.

This research focuses on investigating the impact of non-coding RNAs on stem cell biology and differentiation processes. We found that miR-317 plays a role in germline stem cell progeny differentiation. miR-317 and its neighbor, the lncRNA Peony, originate and are co-expressed from a singular polycistronic non-coding RNA locus. Alternative polyadenylation is implicated in regulation of their differential expression. While the increased expression of the lncRNA Peony results in the disruption of the muscle sheath covering the testis, the absence of miR-317 leads to the emergence of germline tumors in young flies. The deficiency of miR-317 increases Notch signaling activity in the somatic cyst cells, which drives germline tumorigenesis. Germline tumors also arise from upregulation of several predicted targets of miR-317, among which are regulators of the Notch pathway. This implicates miR-317 as a novel tumor suppressor that modulates Notch signaling strength.

Effect of Epigenetic Inhibitors on Adipogenesis in Human Periodontal Ligament Stem Cells

Histone H3 lysine 9 acetylation is a crucial epigenetic mark in mesenchymal stem cell differentiation processes like adipogenesis. This study aimed to evaluate the effect of epigenetic inhibitors on adipogenesis in human periodontal ligament stem cells, using histone deacetylase inhibitors such as valproic acid and trichostatin A. The cells were treated independently with 8 mM valproic acid and 100 nM trichostatin A for 72 hours to inhibit class I histone deacetylases. Subsequently, the cells underwent adipogenic induction for 28 days. Morphology and intracellular lipid deposition were analyzed using Oil-Red oil staining. Protein extracts were prepared on days 0 and 28 to analyze histone H3 lysine 9 acetylation levels via Western blot. Our results demonstrated that cells treated with either VPA or TSA showed, on average, a 1.275-fold increase in lipid deposition during the adipogenic process, with no significant differences between the treatments compared to the control group. Further, by day 28, histone H3 lysine 9 acetylation levels were 1.43 times higher in cells treated with valproic acid and 2.52 times higher in those treated with trichostatin A.These findings suggest that the inhibitors could have differential effects on chromatin remodeling in different regions by increasing acetylation, contributing indistinctly to lipid deposition due to the greater expression of genes associated with the adipogenic phenotype.

AMAR-seq: Automated Multimodal Sequencing of DNA Methylation, Chromatin Accessibility, and RNA Expression with Single-Cell Resolution.

Parallel single-cell multimodal sequencing is the most intuitive and precise tool for cellular status research. In this study, we propose AMAR-seq to automate methylation, chromatin accessibility, and RNA expression coanalysis with single-cell precision. We validated the accuracy and robustness of AMAR-seq in comparison with standard single-omics methods. The high gene detection rate and genome coverage of AMAR-seq enabled us to establish a genome-wide gene expression regulatory atlas and triple-omics landscape with single base resolution and implement single-cell copy number variation analysis. Applying AMAR-seq to investigate the process of mouse embryonic stem cell differentiation, we revealed the dynamic coupling of the epigenome and transcriptome, which may contribute to unraveling the molecular mechanisms of early embryonic development. Collectively, we propose AMAR-seq for the in-depth and accurate establishment of single-cell multiomics regulatory patterns in a cost-effective, efficient, and automated manner, paving the way for insightful dissection of complex life processes.

NJGCG: A node-based joint Gaussian copula graphical model for gene networks inference across multiple states

Inferring the interactions between genes is essential for understanding the mechanisms underlying biological processes. Gene networks will change along with the change of environment and state. The accumulation of gene expression data from multiple states makes it possible to estimate the gene networks in various states based on computational methods. However, most existing gene network inference methods focus on estimating a gene network from a single state, ignoring the similarities between networks in different but related states. Moreover, in addition to individual edges, similarities and differences between different networks may also be driven by hub genes. But existing network inference methods rarely consider hub genes, which affects the accuracy of network estimation. In this paper, we propose a novel node-based joint Gaussian copula graphical (NJGCG) model to infer multiple gene networks from gene expression data containing heterogeneous samples jointly. Our model can handle various gene expression data with missing values. Furthermore, a tree-structured group lasso penalty is designed to identify the common and specific hub genes in different gene networks. Simulation studies show that our proposed method outperforms other compared methods in all cases. We also apply NJGCG to infer the gene networks for different stages of differentiation in mouse embryonic stem cells and different subtypes of breast cancer, and explore changes in gene networks across different stages of differentiation or different subtypes of breast cancer. The common and specific hub genes in the estimated gene networks are closely related to stem cell differentiation processes and heterogeneity within breast cancers.

Vasoactive intestinal peptide exerts an osteoinductive effect in human mesenchymal stem cells.

Several neuropeptides present in bone tissues, produced by nerve fibers and bone cells, have been reported to play a role in regulating the fine-tuning of osteoblast and osteoclast functions to maintain bone homeostasis. This study aims to characterize the influence of the neuropeptide vasoactive intestinal peptide (VIP) on the differentiation process of human mesenchymal stem cells (MSCs) into osteoblasts and on their anabolic function. We describe the mRNA and protein expression profile of VIP and its receptors in MSCs as they differentiate into osteoblasts, suggesting the presence of an autocrine signaling pathway in these cells. Our findings reveal that VIP enhances the expression of early osteoblast markers in MSCs under osteogenic differentiation and favors both bone matrix formation and proper cytoskeletal reorganization. Finally, our data suggest that VIP could be exerting a direct modulatory role on the osteoblast to osteoclast signaling by downregulating the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio. These results highlight the potential of VIP as an osteoinductive differentiation factor, emerging as a key molecule in the maintenance of human bone homeostasis.

Potential probiotic Lactobacillus delbrueckii subsp. lactis KUMS-Y33 suppresses adipogenesis and promotes osteogenesis in human adipose-derived mesenchymal stem cell

Today, probiotics are considered to be living microorganisms whose consumption has a certain number of beneficial effects on the consumer. The present study aimed to investigate the effect of a new probiotic extract (Lactobacillus delbrueckii subsp. lactis KUMS Y33) on the differentiation process of human adipose-derived stem cells (hADSCs) into adipocytes and osteocytes and, as a result, clarify its role in the prevention and treatment of bone age disease. Several bacteria were isolated from traditional yogurt. They were evaluated to characterize the probiotic’s activity. Then, the isolated hADSCs were treated with the probiotic extract, and then osteogenesis and adipogenesis were induced. To evaluate the differentiation process, oil red O and alizarin red staining, a triglyceride content assay, an alkaline phosphatase (ALP) activity assay, as well as real-time PCR and western blot analysis of osteocyte- and adipocyte-specific genes, were performed. Ultimately, the new strain was sequenced and registered on NBCI. In the probiotic-treated group, the triglyceride content and the gene expression and protein levels of C/EBP-α and PPAR-γ2 (adipocyte-specific markers) were significantly decreased compared to the control group (P < 0.05), indicating an inhibited adipogenesis process. Furthermore, the probiotic extract caused a significant increase in the ALP activity, the expression levels of RUNX2 and osteocalcin, and the protein levels of collagen I and FGF-23 (osteocyte-specific markers) in comparison to the control group (P < 0.05), indicating an enhanced osteogenesis process. According to the results of the present study, the probiotic extract inhibits adipogenesis and significantly increases osteogenesis, suggesting a positive role in the prevention and treatment of osteoporosis and opening a new aspect for future in-vivo study.

PAI-1 transfected-conditioned media promotes osteogenic differentiation of hBMSCs.

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/β-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Rapid and accurate identification of stem cell differentiation stages via SERS and convolutional neural networks

Monitoring the transition of cell states during induced pluripotent stem cell (iPSC) differentiation is crucial for clinical medicine and basic research. However, both identification category and prediction accuracy need further improvement. Here, we propose a method combining surface-enhanced Raman spectroscopy (SERS) with convolutional neural networks (CNN) to precisely identify and distinguish cell states during stem cell differentiation. First, mitochondria-targeted probes were synthesized by combining AuNRs and mitochondrial localization signal (MLS) peptides to obtain effective and stable SERS spectra signals at various stages of cell differentiation. Then, the SERS spectra served as input datasets, and their distinctive features were learned and distinguished by CNN. As a result, rapid and accurate identification of six different cell states, including the embryoid body (EB) stage, was successfully achieved throughout the stem cell differentiation process with an impressive prediction accuracy of 98.5%. Furthermore, the impact of different spectral feature peaks on the identification results was investigated, which provides a valuable reference for selecting appropriate spectral bands to identify cell states. This is also beneficial for shortening the spectral acquisition region to enhance spectral acquisition speed. These results suggest the potential for SERS-CNN models in quality monitoring of stem cells, advancing the practical applications of stem cells.

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Decoding the universal human chromatin landscape through teratoma-based profiling.

Teratoma formation is key for evaluating differentiation of human pluripotent stem cells into embryonic germ layers and serves as a model for understanding stem cell differentiation and developmental processes. Its potential for insights into epigenome and transcriptome profiling is significant. This study integrates the analysis of the epigenome and transcriptome of hESC-generated teratomas, comparing transcriptomes between hESCs and teratomas. It employs cell type-specific expression patterns from single-cell data to deconvolve RNA-Seq data and identify cell types within teratomas. Our results provide a catalog of activating and repressive histone modifications, while also elucidating distinctive features of chromatin states. Construction of an epigenetic signature matrix enabled the quantification of diverse cell populations in teratomas and enhanced the ability to unravel the epigenetic landscape in heterogeneous tissue contexts. This study also includes a single cell multiome atlas of expression (scRNA-Seq) and chromatin accessibility (scATAC-Seq) of human teratomas, further revealing the complexity of these tissues. A histology-based digital staining tool further complemented the annotation of cell types in teratomas, enhancing our understanding of their cellular composition. This research is a valuable resource for examining teratoma epigenomic and transcriptomic landscapes and serves as a model for epigenetic data comparison.

Dynamic changes in LINC00458/HBL1 lncRNA expression during hiPSC differentiation to cardiomyocytes

Long non-coding RNAs (lncRNAs) constitute the largest and most diverse class of non-coding RNAs. They localize to the nucleus, cytoplasm, or both compartments, and regulate gene expression through various mechanisms at multiple levels. LncRNAs tend to evolve faster and present higher tissue- and developmental stage-specific expression than protein-coding genes. Initially considered byproducts of erroneous transcription without biological function, lncRNAs are now recognized for their involvement in numerous biological processes, such as immune response, apoptosis, pluripotency, reprogramming, and differentiation. In this study, we focused on Heart Brake lncRNA 1 (HBL1), a lncRNA recently reported to modulate the process of pluripotent stem cell differentiation toward cardiomyocytes. We employed RT-qPCR and high-resolution RNA FISH to monitor the expression and localization of HBL1 during the differentiation progression. Our findings indicate a significant increase in HBL1 expression during mesodermal and cardiac mesodermal stages, preceding an anticipated decrease in differentiated cells. We detected the RNA in discrete foci in both the nucleus and in the cytoplasm. In the latter compartment, we observed colocalization of HBL1 with Y-box binding protein 1 (YB-1), which likely results from an interaction between the RNA and the protein, as the two were found to be coimmunoprecipitated in RNP-IP experiments. Finally, we provide evidence that HBL1, initially reported as an independent lncRNA gene, is part of the LINC00458 (also known as lncRNA-ES3 or ES3) gene, forming the last exon of some LINC00458 splice isoforms.

The Bio-inspired Changeable Production System – A Stem Cell Approach

Volatility can be addressed through changeability. A new bio-inspired approach based on the stem cell differentiation process is presented to meet the needs of a changeable production system. Stem cells possess the property of a raw product that can be processed into various final products. These products are manufactured based on their demand in the same production system with the same production resources. This paper aims to translate the production system from biology to the real world. It does so by translating molecules and their biological mechanisms into production resources, information, and transportation pathways. The paper describes eleven different change scenarios based on Nyhuis’ three-axis changeability model, which correspond to change pathways in the stem cell system and the production system. This model has high potential for use in industry to achieve a changeable production.

TMEM135 maintains the equilibrium of osteogenesis and adipogenesis by regulating mitochondrial dynamics

BackgroundDisturbance in the differentiation process of bone marrow mesenchymal stem cells (BMSCs) leads to osteoporosis. Mitochondrial dynamics plays a pivotal role in the metabolism and differentiation of BMSCs. However, the mechanisms underlying mitochondrial dynamics and their impact on the differentiation equilibrium of BMSCs remain unclear. MethodsWe investigated the mitochondrial morphology and markers related to mitochondrial dynamics during BMSCs osteogenic and adipogenic differentiation. Bioinformatics was used to screen potential genes regulating BMSCs differentiation through mitochondrial dynamics. Subsequently, we evaluated the impact of Transmembrane protein 135 (TMEM135) deficiency on bone homeostasis by comparing Tmem135 knockout mice with their littermates. The mechanism of TMEM135 in mitochondrial dynamics and BMSCs differentiation was also investigated in vivo and in vitro. ResultsDistinct changes in mitochondrial morphology were observed between osteogenic and adipogenic differentiation of BMSCs, manifesting as fission in the late stage of osteogenesis and fusion in adipogenesis. Additionally, we revealed that TMEM135, a modulator of mitochondrial dynamics, played a functional role in regulating the equilibrium between adipogenesis and osteogenesis. The TMEM135 deficiency impaired mitochondrial fission and disrupted crucial mitochondrial energy metabolism during osteogenesis. Tmem135 knockout mice showed osteoporotic phenotype, characterized by reduced osteogenesis and increased adipogenesis. Mechanistically, TMEM135 maintained intracellular calcium ion homeostasis and facilitated the dephosphorylation of dynamic-related protein 1 at Serine 637 in BMSCs. ConclusionsOur findings underscore the significant role of TMEM135 as a modulator in orchestrating the differentiation trajectory of BMSCs and promoting a shift in mitochondrial dynamics toward fission. This ultimately contributes to the osteogenesis process. This work has provided promising biological targets for the treatment of osteoporosis.